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IOSR Journal of Environmental Science, Toxicology and Food Technology (IOSR-JESTFT)

e-ISSN: 2319-2402,p- ISSN: 2319-2399.Volume 8, Issue 12 Ver. III (Dec. 2014), PP 58-64
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Enzymatic Decolorization of Malachite Green Dye by a Newly


Isolated Bacillus Cereus Strain wwcp1
Wycliffe Chisutia Wanyonyi*1, John Mmari Onyari1, Paul Mwanza Shiundu2,
Francis Jackim Mulaa3
1

Department of Chemistry, College of Biological and Physical Sciences,University of Nairobi, P.O. Box 30197 00100, Nairobi, Kenya.
2
Department of Chemical Sciences and Technology, The Technical University of Kenya, Haile Selassie Avenue,
P.O. Box 52428 - 00200, Nairobi, Kenya
3
Department of Biochemistry, College of Health Sciences, University of Nairobi, P.O. Box 30197 - 00100,
Nairobi, Kenya.

Abstract: Enzymatic decolorization of Malachite Green (MG) dye was studied using crude enzyme from a
newly isolated Bacillus cereus strain wwcp1. 98% decolorization efficiency was achieved within 24 hours using
an initial dye concentration of 1.0 x 10-5M. Batch experimental results revealed that the decolorization process
was highly dependent on contact time, initial MG concentration, aqueous solution temperature and pH.
Biodegradation of MG dye was monitored spectrophotometrically and metabolites confirmed by thin layer
chromatography (TLC). The comparison of TLC chromatograms before and after decolorization confirmed that
crude protease enzyme had the ability to degrade MG dye. The results provide evidence that the crude enzyme
from Bacillus cereus strain wwcp1 is an effective and potential candidate for industrial wastewater treatment.
Keywords: Decolorization, Malachite Green Dye, Bacillus cereus strain wwcp1, Biodegradation, Enzyme.

I.

Introduction

Water pollution due to release of industrial wastewater has become a serious environmental problem in
almost every industry using dyes to color products. Wastewater from textile, paper, leather, cosmetic, food, and
plastic industries often contains synthetic dyes that are toxic, mutagenic, and carcinogenic [1, 2]. The strong
color of industrial wastewaters containing dyes even at a small concentration has a huge impact on the aquatic
environment due to their turbidity, increased chemical oxygen demand (COD), and reducing light penetration,
which has adverse effects on photosynthetic phenomena. Among many classes of synthetic dyes used in the
industries, triphenyl methane group of dyes such as malachite green and crystal violet constitute a major and
versatile group that play a predominant role in almost every type of application [3].
Malachite oxalate green dye (MG) [(C23H25N2)(C2HO4)]2C2H2O4], (Bis[[4-[4-(dimethylamino)
benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium] oxalate, dioxalate; N,N,N',N'-Tetramethyl4,4'-diaminotriphenylcarbenium oxalate) MW 929.03g (Figure 1) has been found to be highly toxic to
mammalian cells; promote hepatic tumor formation in rodents and cause reproductive abnormalities in rabbits
and fish [4]. It was nominated by the Food and Drug Administration as a priority chemical for carcinogenicity
testing by the National Toxicology Programme of the USA [5]. The need to maintain a cleaner environment for
the survival of both aquatic and terrestrial lives has forced many governments to established environmental
restrictions with regard to the quality of colored effluents forcing dye houses to decolorize their effluents before
discharging. Treatment of industrial wastewater containing dyes is an essential, but difficult process because
dyes usually have a complex aromatic structure which makes them resistant to biodegradation by conventional
biological treatments.
Several methods have been developed to remove synthetic dyes from wastewaters in order to decrease
their impact on the environment [6, 7]. Conventional physicochemical methods such as adsorption with
activated carbon, coagulation, precipitation, solvent extraction, membrane filtration, chemical oxidation,
ozonation, and flocculation have been used to treat dye containing effluents; however, most of these methods
are expensive or require additional chemicals [8]. The decolorization of dye in wastewater using microbial
enzymes has been a subject of many studies in recent years due to their low cost of production and efficient
application. Compared to physicochemical treatment methods, the enzymatic treatment of dyes have low energy
cost and are more ecofriendly process although not commonly used in the textile industries [9].

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Enzymatic Decolorization of Malachite Green Dye by a Newly Isolated Bacillus Cereus Strain wwcp1
CH3

N
H3C

OH
O
2
OH
HO

N
H3C

CH3
2

Figure 1: Structure of Malachite Green Oxalate (MG) dye


Extremophile like alkaliphiles bacteria exhibit the ability to grow at the extremely harsh environmental
conditions such as high pH and temperature, high levels of salinity or salt, and pressure which critically
influence their growth. Products of industrial importance from alkaliphiles have been commercialized, the most
successful of which have been in the detergent and food industries [10]. However, relatively few studies have
been reported on the application of alkaline enzyme in decolorization of organic dyes. The aim of present study
is to investigate the ability of crude protease enzyme from newly isolated Bacillus cereus strain wwcp1 in
decolorizing MG dye. Batch experiment were contacted to investigate and optimize the effects of contact time,
pH, initial MG concentration, aqueous solution temperature on the decolorization of MG dye by crude protease
enzyme. The resultant metabolites were analyzed by thin layer chromatography.

II.

Materials And Methods

A cationic basic dye, malachite green oxalate, was obtained from Loba-Austria and used without
further purification. MG solution was prepared from stock solution of 1.010-4M (92.903 mg/L) by dilution. All
solutions were prepared in double distilled water and pH adjusted by adding either 0.1 M HCl or NaOH.
1.1 Microorganism Isolation and enzyme production
Dye-decolorizing bacteria were isolated from mud water samples obtained from Lake Bogoria. One
gram of the contaminated soil sample was weighed aseptically into 100 ml of sterile distilled water, agitated for
45 minutes on a shaker and 0.2 ml spread on casein agar plates before incubating at 37C for 2 days. Protease
producing strains were selected by spotting the bacterial cultures on culturing medium containing Reese Agar
medium [11]. Four positive colonies were identified by the formation of zone of clearance around the colony.
The zone was made clearer by flooding the plates with a solution of 5% Trichloroacetic acid (TCA). The
diameter of the bacterial colony and the total zone of enzyme activity including the growth diameter were
measured in each case. Bacillus cereus strain wwcp (GenBank accession No. KM201428) exhibited maximum
clear zone around the colony indicating high protease activity and thus selected for enzyme production. The
medium for crude enzyme production contained 0.5 % casein and 0.25% glucose. 500mL Erlenmeyer flasks
containing 100mL of media plugged in cotton and aluminum foil was sterilized in autoclave at 121C (15 lb) for
15 min and after cooling the flask was inoculated with 5% over night grown seed bacterial culture. The
inoculated medium was incubated at 45C on a rotary shaker (140 rpm) incubator. After 72 hours, the culture
medium was centrifuged at 5000 rpm and 4C for 15 min to obtain the crude extract which served as crude
enzyme source.
1.2 Assay of Enzyme Activity
Protease activity in the crude enzyme was assayed by the modified Tsuchida procedure [12] using 1%
casein as substrate. Casein solution (1.0 ml) was mixed with an equal volume of crude protease enzyme solution
and incubated at 45C for 10 min. The reaction was terminated by the addition of 4 ml of 10% (w/v) chilled
trichloroacetic acid and reaction mixture allowed to stand in ice for 20 min to precipitate the insoluble proteins.
The mixture was centrifuged to obtain the supernatant. 5ml of 0.4M Na2CO3 and 1 ml of one fold diluted Folin
ciocalteau reagent was added to the supernatant which was further incubated for 30 minutes to develop the
color. The absorbance was measured against an appropriate blank at 660 nm using a UV-VIS
spectrophotometer.
1.3 Effect of pH on crude proteases enzyme activity
In order to determine the optimum pH for protease activity, the crude enzyme was assayed in 1%
Casein (w/v) as substrate dissolved in buffers of different pH ranging from 4 to 12. The pH was adjusted using
the following buffer systems at 0.1M concentration: acetate (pH 4, 5), sodium phosphate buffer (pH 6, 7), Tris
HC1 buffer (pH 8) and glycine-NaOH (pH 9 - 12). Reaction mixtures were incubated at 45C for 30 min before
measuring the enzyme activity.
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Enzymatic Decolorization of Malachite Green Dye by a Newly Isolated Bacillus Cereus Strain wwcp1
1.4 Decolorization Assay
1.4.1
Determination of wavelength of maximum absorbance (max) for Malachite green
A 1.0 x 10-5M solution of MG dye was prepared from the stock solution of 1.0 x 10 -4M. A sample of
dye solution in a cuvette was placed in the UV-VIS spectrophotometer that had been set to the absorbance
mode. Starting from the lower wavelength (400 nm), the wavelength was adjusted and the wavelength together
with the corresponding absorbance recorded over the entire wavelength range. The spectrophotometer was then
set to the maximum wavelength value (617 nm) for all the absorbance measurements thereafter.
1.4.2

Determination of the effect of contact time on MG dye decolorization


10ml of crude protease enzyme was mixed with 40 ml of 1.0 x 10 -5M MG dye solution at room
temperature in 100ml conical flask on a Thermolyne Orbital shaker at 150 rpm. The pH of the crude enzyme
and dye solution was adjusted to pH 8.5. After a time interval of 10 minutes, aliquots from the reaction mixture
were analyzed for residual MG concentrations using a UVVis spectrophotometer (UV-min 1240 SHIMADZU)
monitoring changes in absorbance at 617 nm. Control tests were conducted where crude protease enzyme had
been replaced by deionized water. All experiments were performed in triplicate and results expressed as the
mean values.
1.4.3

Determination of the effect of initial MG dye concentration on decolorization


The effect of initial dye concentration was investigated at room temperature (25 C) and pH 8.5. 40 ml
of MG dye of concentrations at various increasing concentrations ranging from 1.0 x 10 -6M, to 1.0 x 10-5M was
placed in 100ml conical flask and 10 ml of crude protease enzyme added and placed on an orbital shaker.
Samples were collected from the flask at various time intervals and analyzed for residual MG concentration. The
rate of decolorization was expressed as the percentage decrease in absorbance at the peak wavelength.
1.4.4
Determination of the effect of temperature on MG dye decolorization
The effect of temperature on MG dye decolorization was investigated at temperature range of 25 - 70C and pH
8.5. 40 ml of MG dye of initial concentration of 1.0 x 10-5M was mixed with 10 ml of crude protease enzyme
both pre equilibrated at the study temperature for 30 minutes before mixing in 100ml conical flask. Absorbance
reading was taken at an interval of 10 minutes till equilibrium was attained.
1.4.5
Determination of the effect of pH on MG dye decolorization
The effect of pH was investigated at room temperature (25C) and a pH range of 4-11 for 5 and 12 hours. 10 ml
of crude enzyme at appropriate pH was mixed with 40 ml of 1.0 x 10-5M MG dye solution in 100ml conical
flask. The rate of decolorization was expressed as the percentage decrease in absorbance at the peak wavelength.
1.4.6
Assay of metabolites formed from the biodegradation
After complete decolorization, the metabolites from biodegraded products were extracted with equal volume of
ethyl acetate (25: 25 ml). The extracts were dried over anhydrous Na 2SO4 and evaporated to dryness in rotary
evaporator. The dry crystals were placed in 25 ml conical flask and 5ml of methanol added to dissolve the
metabolites for TLC analysis. Metabolite formation was examined by thin layer chromatography (TLC) using
silica gel activated in chloroform. The solvent system used was n-propanol: ethyl acetate: acetic acid: distilled
water (6:1:1:2 v/v). The separated products were visualized in iodine chamber.

II.

Results And Discussion

2.1 Effect of pH on crude proteases enzyme activity


The crude protease enzyme from Bacillus Cereus Strain wwcp1 showed good protease activity over a
broader pH range in alkaline media. The optimum pH was found to be 11 (Figure. 2), indicating that the enzyme
is alkaline protease. Similar results were also observed in the protease produced by Aspergillus clavatus [13].

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Enzymatic Decolorization of Malachite Green Dye by a Newly Isolated Bacillus Cereus Strain wwcp1

Absorbance at 660 nm

0.7
0.6
0.5

0.4
0.3
0.2

0.1
0

8
pH

10

11

12

13

Figure 2. Effect of pH on Bacillus Cereus Strain wwcp1 protease activity


2.2 Determination of the effect of contact time on MG decolorization
The effect of contact time on MG dye decolorization by crude enzyme was examined by varying the
time of incubation and the results presented in Figure 3. The results show that dye decolorization was rapid
within the first 4 hours with approximately 82 % dye decolorization. The change in percentage decolorization
after the 4th hour became relatively gradual attaining equilibrium at the 20th hour with 97.5 % dye decolorization.
This observation suggested that the initial four hours was significant for dyes decolorization but quite slow after
fourth hour which may be probably due to products inhibition. These results were in agreement with earlier
published work of decolorization of textile dyes [14].
120

% Dye decolization

100
80
60
40
20
0
0

10

Time15
in hours

20

25

30

Figure 3: Effect of contact time on MG dye decolorization


2.3 Effect of pH on Malachite Green dye Decolorization
Enzymes are greatly affected by variation in pH. The effect of pH on decolorization of MG dye by
crude protease enzyme was investigated at various pH values and results presented in Figure 4. It can be inferred
that at a pH below 5, there was significantly very low decolorization rate but when the pH increased above 6.0,
the rate of decolorization rapidly increased from 21% at pH 6 to 98% at pH 11. The result shows that the
optimum pH for efficient MG dye decolorization was between pH 9 and 11. These results obviously present an
advantage from industrial application point of view since most of dye effluents are characterized by alkaline pH
under which crude protease enzyme works optimally. These findings are consistent with related studies done on
decolorization of textile azo dyes by newly isolated halophilic and halotolerant bacteria [15].

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Enzymatic Decolorization of Malachite Green Dye by a Newly Isolated Bacillus Cereus Strain wwcp1

Figure 4: Effect of pH on the decolorization of MG dye


2.4 Effect of Temperature on Malachite Green dye Decolorization
Temperature plays an important role in enzyme activity. Effect of temperature on decolorization of MG
dyes was investigated at temperature range of 25 - 70C and results presented in Figure 5. The results show that
the rate of MG dye decolorization increased with increase in temperature between 25 C, and 40C with
decolorization efficiency increasing with increase of incubation time. The optimum temperature for crude
protease enzymes was found to be 40C with 83% decolorization efficiency after 4 hours. At elevated
temperature above 50C, the rate of dye decolorization increased during the first 20 minutes but after one hour,
percent dye decolorization remarkably decreased. This can be attributed to the denaturation of enzyme and
thermal inactivation of enzyme under the operating temperature.

Figure 5: Effect of temperature on the decolorization of MG dye


2.5 Effect of initial Malachite Green dye concentration on decolorization
The decolorization of MG was studied at various increasing concentration of dye from 1.0 x 10 -6M, to
1.0 x 10-5M and the results presented in Figure 6. It could be inferred from the results that the rate of
decolorization decreased with the increase in initial MG dye concentration. However, the crude protease enzyme
was able to decolorize higher concentration in the range of 83100% during 24 h incubation period. The
decrease in decolorization efficiency at high concentration might be due to the toxic effect of dye through the
inhibition of metabolic activities. Similar results were observed by Murugesan et al [16] who investigated the
decolorization of reactive dyes by a thermostable laccase produced by Ganoderma lucidum in solid state culture
and indicated that decolorization of RB-5 and RBBR dyes decreased with increasing dye concentration.

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Enzymatic Decolorization of Malachite Green Dye by a Newly Isolated Bacillus Cereus Strain wwcp1

Figure 6: Effect of initial Malachite Green dye concentration on decolorization


2.6 Analysis of MG degraded metabolites
The metabolites produced during the biodegradation of malachite green dye were analyzed by thin
layer chromatography and the results presented in Figure 7a and 7b. The comparison of TLC chromatograms
before and after decolorization by crude protease enzyme in iodine chamber showed the appearance of three
additional bands (M1, M2, and M3 of retardation factor (Rf) values of 0.96, 0.92 and 0.90 respectively (Figure
7b) as compared to control Rf value of 0.87 which might have been originated from the degraded dye
metabolites. TLC results suggested that crude protease enzyme was able to degrade MG dye giving rise to three
main metabolites which accounted for color disappearance.

b
M1
M2
M3
MG

Ce

MG.d

MG

MG.d

Figure 7. TLC Chromatograms of MG dye before and after degradation by crude protease enzyme (a) column
1,2 & 3 represent negative control with enzyme alone (Ce), degraded Malachite green dye (MG.d) and positive
control with untreated Malachite green dye (MG)respectively; (b) Three metabolites (M1, M2, & M3) of
degraded Malachite green dye.

III.

Conclusion

Application of conventional physicochemical waste water treatment requires enormous cost and
continuous input of chemicals which becomes uneconomical and causes further environmental damage. In the
present study, our results clearly demonstrated that crude protease enzyme isolate from Bacillus Cereus Strain
wwcp1 exhibit a novel alkaline protease properties with ability to decolorize and degrade MG dye. Taking these
results into consideration, it can be concluded that newly isolated Bacillus Cereus Strain wwcp1 can be
exploited for bioremediation of triphenyl methane group of dyes as a cost effective alternative technology for
dye removal in wastewater treatment processes.

Acknowledgements
We are thankful to National Commission for Science, Technology and Innovation (NACOSTI)
research endowment fund (Project no. NCST/ST&I/RCD/4TH CALLPhD/140), World Federation of Scientists
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Enzymatic Decolorization of Malachite Green Dye by a Newly Isolated Bacillus Cereus Strain wwcp1
(WFS) Scholarship through the International Centre for Insect Physiology and Ecology (ICIPE) and Department
of Chemistry, University of Nairobi for supporting and providing the infrastructure facilities for this study.

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